We investigated roles of α4 integrins during hematopoiesis using mutant and chimeric mice. Yolk sac erythropoiesis and migration of hematopoietic progenitors to fetal liver, spleen, and bone marrow can occur without α4 integrins. Although terminal differentiation of these progenitors is possible without α4 integrins, these receptors are essential to maintain normal hematopoiesis in fetal liver, spleen, and bone marrow microenvironments. Moreover, α4-deficient erythroid progenitors and pre-B cells neither transmigrate beneath the stroma nor expand properly in vitro. In contrast, α4-null cells migrate and differentiate efficiently into T lymphocytes within the thymus. In summary, α4 integrins are essential for normal development of all hematopoietic lineages in fetal liver, bone marrow, and spleen, likely by regulating the proliferation/differentiation balance of hematopoietic progenitors.